Evidence that human haematopoietic stem cells (HSC) do not reside within the CD34+KIT- cell population

Contradictory reports are published concerning the c-kit receptor (KIT) expression on human haematopoietic stem cells (HHSC). Therefore, the aim of this study was to reevaluate the expression of KIT on human early haematopoietic cells, and to study the distribution of HHSC among bone marrow mononuclear KIT+ and KIT- cells. First, we found that the detection sensitivity of the KIT expression on human HEL cells as well as CD34+ depends on the type of fluorochrome employed for the immunostaining (Cy5 > PE > FITC). Based on this observation, in our strategy for isolating human HHSC we employed a Cy-5 conjugated alpha-KIT MoAbs, which stained CD34+ cells in our preliminary studies the brightest. Accordingly, we labeled human BMMNC with PE-alpha-CD34 and Cy5-alpha-KIT MoAbs and subsequently sorted various subsets of labeled cells (CD34+KIT+, CD34+KIT- and CD34-KIT-). Cells sorted by FACS were then evaluated for their ability to engraft in the immunodeficient SCID mice model. We report here that only CD34+KIT+ cells, but not CD34+KIT- or CD34-KIT- were able to establish a human-murine haematopoietic chimerism in these animals. We found that SCID mice transplanted with CD34+ KIT+ cells, possessed approximately 5-11% of mononuclear cells, which expressed human CD45 antigen 4-5 weeks after transplantation in their bone marrow and, more importantly, early human haematopoietic progenitors from the myeloid and B-lymphoid lineages. Based on these results we conclude that KIT (CD117) is a very useful marker for identifying HHSC, and that HHSC, at least in our hands, are found in the KIT+ population of CD34+ cells.